Lysocellin is a divalent polyether antibiotic produced by culturing Streptomyces type microorganisms. Polyether antibiotics as a class are reviewed in Westley, Adv. Appl. Microbiology. 22:177-223 (1977). Westley classified lysocellin in Class 2a; Class 2a antibiotics have a generally linear configuration, may contain from about two to about three tetrahydropyran and/or -furan structures, up to about three tetrahydropyran and/or -furan structures, up to about three total ring structures and no nitrogen atoms. The isolation and purification of polyether antibiotics using extraction methods have been extensively reviewed in Hamill et al., "Polyether Antibiotics" pp. 479-520, J. Chromatogr. Lib., Vol. 15. Antibiotics: Isolation, Separation, and Purification, ed. by Weinstein, M. J. and Wagman, G. H. (1978).
Procedures for producing, isolating, and purifying lysocellin are known in the art. Lysocellin is generally produced by fermenting a nutrient-containing liquid fermentation medium or broth inoculated with a microorganism capable of producing the desired antibiotic. Suitable liquid fermentation media are generally aqueous dispersions containing sources of assimilable nitrogen and carbon as is known in the art. The fermentation media can also contain a variety of optional ingredients, if desired, such as for example, pH adjustment agents, buffers, trace minerals, antifoam agents, and the like.
Known methods for recovering lysocellin from fermentation broths generally involve multi-stage organic solvent extractions and related filtration, chromatography, concentration, and crystallization operations. The procedure to isolate and purify lysocellin was first described in Ebata et al., J. Antibiotics, 28:118-121 (1975). Ebata used a multi-step organic extraction process which incorporated acetone, n-butanol, methanol, and the like. U.S. Pat. No. 4,033,823 describes an extraction process for recovering lysocellin which incorporates ethyl acetate, acetonitrile, hexane and methanol. U.S. Pat. No. 4,478,935 describes various purified manganese-containing antibiotic complexes extracted from the dried biomass using suitable organic solvents followed by crystallization or precipitation of the complexes. All of these processes follow a rather standard approach in which fermentation broths are subjected to organic solvent extraction to recover the lysocellin as a crystalline solid.
Production of lysocellin by these procedures produces, as a general rule, relatively pure lysocellin solids which can be used commercially. However, some fermentation batches produce lysocellin solids which contain large amounts of impurities in the form of fatty acids or fatty acid ester salts formed from undesirable metal cations, particularly fatty acid ester salts formed with metal cations such as magnesium, calcium, iron, and the like. These fatty acid ester salts containing metal ion impurities often result when fermentation broths or nutrients contain high levels of these metal ions, although many other sources can contaminate the broth. The presence of large amounts of these fatty acid or ester salts make the lysocellin solids produced from the lysocellin production process unusable for their intended purpose.
Prior methods for removing these impurities have generally involved organic extraction procedures in which the impurities and lysocellin are separated based upon their differing solubilities in various organic solvents. The problem has been that, as with all extraction procedures, a portion of the solubilized lysocellin is lost during the solubilization and extraction procedure. The lysocellin lost during the extraction decreases the yield in the purification procedure. More often, because of these difficulties, lysocellin solids containing large amounts of impurities are merely discarded.
In addition, because the sodium form of lysocellin is very stable in a variety of environments, sodium lysocellin is more suitable than other salts for animal feed applications. However, since calcium, magnesium, iron, and other metal ions may exhibit greater affinity to the divalent lysocellin ionophore than sodium for lysocellin concentrations higher than 1.5 g/L (Mitani et al., J. Antibiotics, 30, 186-88, 1977), it is difficult to isolate lysocellin in the sodium form once other salts have been formed during fermentation.
There has not been an effective process for separating lysocellin from these impurities, fatty acids and fatty acid ester salts, in which the lysocellin is not solubilized in an extraction procedure. A procedure in which the lysocellin remains in a solid form, not solubilized, would avoid the accompanying loss of lysocellin resulting from the solubilization in the purification procedure. A method is, therefore, needed which can purify lysocellin solids by solubilizing and removing fatty acid and fatty acid ester salt impurities while maintaining the lysocellin as a solid.